Using the c-fos enhancer as a model to analyze growth hormone (GH)-promoted gene expression, the roles of CCAAT/enhancer-binding proteins (C/EBPs) in GH-regulated transcription were investigated. In 3T3-F442A fibroblasts stably expressing the c-fos promoter mutated at the C/EBP binding site upstream of luciferase, c-fos promoter activity is stimulated by GH 6-7-fold; wild type c-fos promoter shows only a 2-fold induction by GH. This suggests that C/EBP restrains GH-stimulated expression of c-fos. Electrophoretic mobility shift assays with nuclear extracts from 3T3-F442A cells indicate that GH rapidly (2-5 min) increases binding of C/EBPbeta and C/EBPdelta, to the c-fos C/EBP binding site. Both liver activating protein (LAP) and liver inhibitory protein (LIP), forms of C/EBPbeta, are detected in 3T3-F442A cells by immunoblotting. GH increases the binding of LAP/LAP and LAP/LIP dimers. Overexpression of LIP interferes with GH-promoted reporter expression in CHO cells expressing GH receptors, consistent with the possibility that LIP restrains GH-stimulated c-fos expression. Overexpression of LAP elevates basal luciferase activity but does not influence promoter activation by GH, while overexpressed C/EBPdelta elevates basal promoter activity and enhances the stimulation by GH. GH stimulates the expression of mRNA for C/EBPbeta and -delta and increases levels of C/EBPdelta. Although C/EBPbeta is not detectably altered, GH induces a shift to more rapidly migrating forms of LIP and LAP upon immunoblotting. Treatment of extracts from GH-treated cells with alkaline phosphatase causes a shift of the slower migrating form to the rapidly migrating form, consistent with GH promoting dephosphorylation of LIP and LAP. These studies implicate C/EBPbeta and -delta in GH-regulated gene expression. They also indicate that GH stimulates the binding of C/EBPbeta and -delta to the c-fos promoter and promotes the dephosphorylation of LIP and LAP. These events may contribute to the ability of C/EBPbeta and -delta to regulate GH-stimulated expression of c-fos.